Generation of a cancer-specific immune response toward muc1 and cancer specific muc1 antibodies

ABSTRACT

The present invention provides a method for inducing a cancer specific immune response against MUC1 using an immunogenic glycopeptide. Other aspects of the invention are a pharmaceutical composition comprising the immunogenic glycopeptide and a cancer vaccine comprising the immunogenic glycopeptide. Another aspect is an antibody generated using the immunogenic glycopeptide and the use of said antibody in therapy and diagnosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/040,375, filed on Feb. 10, 2016, which is a continuation of U.S.application Ser. No. 14/539,916, filed on Nov. 12, 2014, which claimsthe benefit of U.S. application Ser. No. 13/864,096, filed on Apr. 16,2013, which claims the benefit of U.S. application Ser. No. 12/444,360,filed on Oct. 22, 2009, which claims the benefit and priority to and isa U.S. National Phase Application of PCT International ApplicationNumber PCT/DK2007/050139, filed on Oct. 4, 2007, designating the UnitedStates of America and published in the English language, which is anInternational Application of and claims the benefit of priority to U.S.Provisional Application No. 60/848,997, filed on Oct. 4, 2006. Thedisclosures of the above-referenced applications are hereby expresslyincorporated by reference in their entireties.

SEQUENCE LISTING IN ELECTRONIC FORMAT

The present application is being filed along with a Sequence Listing inelectronic format. The sequence listing is provided as a file entitled“2018 Jun. 11 Sequence Listing—PLOUG79.001C4”, created Jun. 11, 2018,which is 4 KB in size. The information in the electronic format of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND

The human mucin MUC1 is a polymorphic transmembrane glycoproteinexpressed on the apical surfaces of simple and glandular epithelia. MUC1is highly over-expressed and aberrantly O-glycosylated inadenocarcinomas. The extracellular domain of the mucin contains variablenumber of tandem repeats (25-125) of 20 amino acid residues with fivepotential sites for O-glycosylation. O-glycans are incompletelyprocessed in cancer cells resulting in the expression of thepancarcinoma carbohydrate antigens Tn (GalNAcα1-O-Ser/Thr), STn(NeuAcα2-6GalNAcα1-O-Ser/Thr), and T (Galβ1-3GalNAcα1-O-Ser/Thr). MUC1expressed by breast carcinoma cells carries the short cancer-associatedTn, STn, and T antigens as well as the mono- and disialyl core 1structure (ST, NeuAcα2-3Galβ1-3[NeuAcα2-6]+/-GalNAcα1-O-Ser/Thr) foundwidely in normal cells. In contrast, MUC1 expressed in normal breastepithelial cells generally carry branched core 2 O-glycans(Galβ1-3[GlcNAcβ1-6] GalNAcα1-O-Ser/Thr) with lactosamine extensions.The cell membrane bound mucin MUC1 has long been considered a primetarget for immunotherapeutic intervention. The existence of anti-MUC1antibodies and circulating immune complexes containing MUC1 in breastcancer patients that correlates with improved prognosis, clearlysupports MUC1 as a target. However, stimulation of an effective cellularor humoral immune response to cancer-associated forms of MUC1 inpatients or transgenic animals expressing the human MUC1 gene (usingdefined immunogens as opposed to cell based therapies) have not beenachieved. Strategies for active specific immunotherapy based onpeptide/protein immunogens have so far been limited to unglycosylatedMUC1 tandem repeat peptides of different lengths, conjugated todifferent carriers, or administered with an adjuvant. These strategieshave generally failed to produce effective immune responses to MUC1expressed by cancer cells in hosts where the mucin is expressed as aself antigen.

In the past, a large number of monoclonal antibodies (MAbs) have beenproduced to purified MUC1 and synthetic peptides and glycopeptidesderived from MUC 1. The epitopes of these MAbs have traditionally beendefined by scanning overlapping short peptides, and most of the MAbsdefine epitopes in the heavily O-glycosylated mucin tandem repeatdomain. One large group of MAbs have been raised against human milk fatglobule (HMFG) including HMFG1, 115D8, and SM3, most of which react withan epitope in the PDTR (SEQ ID NO. 4) region of the MUC1 tandem repeatconsidered to be the immunodominant peptide epitope in wild type mice.Only a few MAbs defining tandem repeat epitopes outside the PDTR (SEQ IDNO: 4) region have been reported. One generated against breast cancertissue extract, DF3, is used in the CA 15-3 screening assay incombination with 115D8 and defines the peptide epitope TRPAPGS (SEQ IDNO. 5). Immunization with unglycosylated MUC1 peptide has given rise toa low-affinity monoclonal antibody (BCP9) reactive with the GSTAP (SEQID NO. 3) peptide.

Most MUC1 antibodies react with the peptide backbone but often thebinding is modulated by the presence of glycans. In some cases thepresence of a particular glycan can enhance binding as seen with B27.29,115D8, and VU-2-G7. In other examples glycans can inhibit binding, asseen with SM3 and HMFG1. SM3 was raised against chemicallydeglycosylated HMFG and exhibits high preference for cancer-associatedMUC1—opposed to other MAbs raised against HMFG—because the antibodybinding to the PDTR (SEQ ID NO. 4) region is selectively blocked bylarge branched O-glycans as found in normal breast epithelium (Burchellet al. 2001).

A few antibodies reacting specifically with MUC1 glycoforms have beenreported. One MAb, BW835, was generated by alternating injections ofcancer cell lines MCF-7 and SW-613, and the specificity is reported tobe restricted to the glycopeptide epitope VTSA (SEQ ID NO. 6) where Thris substituted with the T antigen (Galβ1-3GalNAcα1-O-Ser/Thr) (Hanischet al. 1995). The MAb MY.1E12 (Yamamoto et al. 1996) was raised againstHMFG and the epitope maps to the same peptide sequence, but heresialylation of the T structure (ST) enhances reactivity (Takeuchi et al.2002).

Recently, we found that immunization with long Tn- or STn-MUC1 tandemrepeat glycopeptides can override tolerance in humanized MUC1 transgenicBalb/c mice (Sorensen et al. 2006 and example 1 of the presentspecification). The humoral immune response induced with theglycopeptide vaccines was highly specific for the Tn/STn-MUC1 glycoformsand MUC1 expressed by human cancer cells. In order to furthercharacterize immunity to these glycopeptides, we generated monoclonalantibodies that mimic the polyclonal response elicited in MUC1transgenic mice.

SUMMARY OF THE INVENTION

The present invention provides a method for inducing a highlycancer-associated or cancer specific immune response against MUC1 usingan immunogenic glycopeptide. Other aspects of the invention are apharmaceutical composition comprising the immunogenic glycopeptide and acancer vaccine comprising the immunogenic glycopeptide. Another aspectis an antibody generated using the immunogenic glycopeptide and the useof said antibody in therapy and diagnosis.

DETAILED DESCRIPTION OF THE INVENTION Method of Inducing a CancerSpecific Immune Response Toward MUC 1.

Surprisingly, immunization with an immunogenic glycopeptide comprising aglycosylated GSTA (SEQ ID NO. 7) motif has been shown to induce a cancerspecific immune response toward MUC1. E.g. it has been shown thathumoral immunity toward cancer cells can be generated.

When referring to the “immunogenic glycopeptide” herein, what is meantare all the embodiments described below of the immunogenic glycopeptideused for inducing a cancer specific immune response.

Thus, one aspect of the present invention is directed to a method ofinducing a cancer specific immune response toward MUC1 comprisingimmunization of a mammal with an immunogenic glycopeptide comprising aGSTA (SEQ ID NO. 7) motif, wherein said GSTA (SEQ ID NO. 7) motif isO-glycosylated at least at the T-residue or at the S-residue of the GSTA(SEQ ID NO. 7) motif.

Preferably, the mammal is selected from the group consisting of: ahuman, a mouse, a rat, a rabbit, a sheep, a goat, and a dog.

In a preferred embodiment, the immune response toward MUC1 is eitherinnate immunity, humoral immunity, cellular immunity or any combinationshereof.

In another preferred embodiment, MUC1 is aberrantly glycosylated andexpressed on cancer cells. I.e. the immune response is preferentiallydirected toward MUC1 that is aberrantly glycosylated and expressed oncancer cells and to a lesser degree toward MUC1 with a normalglycosylation pattern, e.g. branched core 2-based structures.

As referred to herein, a GSTA (SEQ ID NO. 7) motif is a stretch of fouramino acids, wherein the letters denote the identity of the amino acidswith the one-letter amino acid code.

An O-glycosylation as referred to herein denotes the presence of a sugargroup at the hydroxyl group of the side chain of serine or threonine.

A Tn glycosylation as referred to herein can also be described as(GalNAcα1-O-Ser/Thr), i.e. GalNAcα1 substitution at the side chainhydroxyl of a serine or a threonine.

An STn glycosylation as referred to herein can also be described as(NeuAcα2-6GalNAcα1-O-Ser/Thr), i.e. NeuAcα2-6GalNAcα1 substitution atthe side chain hydroxyl of a serine or a threonine. The sialic acid ofSTn may be O-acetylated at any —OH position.

Preferably, the O-glycosylation of the GSTA (SEQ ID NO. 7) motif iseither an STn glycan or a Tn glycan.

In a preferred embodiment, the S-residue and T-residue of the GSTA (SEQID NO. 7) motif is O-glycosylated at the same time. In this embodiment,the S and T residue may carry the same O-glycosylation or they may carrydifferent O-glycosylations.

Thus, in a preferred embodiment, the O-glycosylation of the S-residueand the T-residue is either STn glycan or Tn glycan.

In another preferred embodiment, the GSTA (SEQ ID NO. 7) motif ispresent in a tandem repeat of 20 amino acid residues, said tandem repeatcomprising five potential sites for O-glycosylation.

As referred to herein, a tandem repeat is a repeated sequence beingfound in a natural protein. A preferred tandem repeat is the tandemrepeat sequence of MUC 1.

In a preferred embodiment, at least 3 of the 5 potential sites forO-glycosylation of the tandem repeat are glycosylated and carryingeither Tn or STn.

In another preferred embodiment, all five potential sites forO-glycosylation are carrying either Tn or STn.

We have demonstrated that the capability of the immunogenic glycopeptideto induce an immune response against the MUC1 protein is dependent onthe degree of glycosylation of the immunogenic glycopeptide. Thus, ahigher degree of glycosylation induces a stronger immune response.However, in some situations, a strong immune response may not be desiredor necessary. E.g. for the generation of hybridoma cells producingantibodies that bind the MUC1 protein or aberrantly glycosylated MUC1protein, the immune response need not necessarily be strong.

In a preferred embodiment, the GSTA (SEQ ID NO. 7) motif is present in atandem repeat, wherein the sequence of the tandem repeat is selectedfrom the group consisting of:

-   -   a) VTSAPDTRPAPGSTAPPAHG (SEQ ID NO.1)    -   b) naturally occurring variants of SEQ ID NO:1 with at least 75%        similarity to SEQ ID NO:1    -   c) artificial variants of SEQ ID NO:1 wherein said artificial        variants are prepared by one or more conservative substitutions        and wherein said artificial variants have at least 75%        similarity to SEQ ID NO:1    -   d) truncated fragments of SEQ ID NO:1 with 1-3 deleted amino        acids

SEQ ID NO:1 is the tandem repeat sequence of MUC 1 as present in humans.

Naturally occurring variants of SEQ ID NO:1 with at least 75% similarityto SEQ ID NO:1 is to be understood as variants of SEQ ID NO:1 that existin nature. In other embodiments, it is preferred that naturallyoccurring variants have a degree of similarity selected from the groupconsisting of 80%, 85%, 90% and 95%.

Artificial variants of SEQ ID NO:1 as referred to herein are variantsthat have been prepared artificially e.g. by genetic engineering orchemical synthesis. Typically, artificial variants will be preparedusing one or more conservative substitutions.

Truncated fragments of SEQ ID NO:1 has been truncated at either theN-terminal end of the peptide, the C-terminal end of the peptide or atboth ends. In a preferred embodiment, the length of the truncation(number of deleted amino acid residues) is selected from the groupconsisting of: 1 residue, 2 residues, 3 residues, 4 residues, 5residues, 6 residues, 7 residues, 8 residues, 9 residues and 10residues. When the truncated fragment is truncated in both ends, theminimum length of the peptide will be selected from the group consistingof: 9 amino acid residues, 10 amino acid residues, 11 amino acidresidues, 12 amino acid residues, 13 amino acid residues, 14 amino acidresidues, 15 amino acid residues and 16 amino acid residues.

In a preferred embodiment, the GSTA (SEQ ID NO. 7) motif is present in atruncated fragment of SEQ ID NO:1 or variants thereof having a degree ofsimilarity of at least 70% relatively to the truncated fragment.

Conservative substitutions as referred to herein are substitutions ofone amino acid residue with another amino acid residue of like charge,size or hydrophobicity.

Preferred conservative substitutions are those wherein one amino acid issubstituted for another within the groups of amino acids indicatedherein below:

-   -   Amino acids having polar side chains (Asp, Glu, Lys, Arg, His,        Asn, Gln, Ser, Thr, Tyr, and Cys,)    -   Amino acids having non-polar side chains (Gly, Ala, Val, Leu,        Ile, Phe, Trp, Pro, and Met)    -   Amino acids having aliphatic side chains (Gly, Ala Val, Leu,        Ile)    -   Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)    -   Amino acids having aromatic side chains (Phe, Tyr, Trp)    -   Amino acids having acidic side chains (Asp, Glu)    -   Amino acids having basic side chains (Lys, Arg, His)    -   Amino acids having amide side chains (Asn, Gln)    -   Amino acids having hydroxy side chains (Ser, Thr)    -   Amino acids having sulfur-containing side chains (Cys, Met),    -   Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser,        Thr)    -   Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and    -   Hydrophobic amino acids (Leu, Ile, Val)

Particular preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Various methods of determining the degree of similarity or the percentsimilarity between two peptides are known. When referring to degree ofsimilarity or percent similarity herein, the following method is used:

The peptides to be compared are aligned optimally. An alignment programmay aid in performing the best alignment. When the two sequences arealigned, a score can be assigned that indicate the degree of similaritybetween the two peptides. Positions with identical amino acid residuesare assigned a score of 1. Positions with conservative substitutions areassigned a score of 0.5. Gaps introduced to optimize the alignment areassigned a score of 0.25. Non-conservative substitutions are assigned ascore of 0. After scoring all positions over the window of comparison,the score is summarized and normalized against the length of the windowof comparison. The normalized value is the percent similarity or degreeof similarity as used herein. Consider e.g. a tandem repeat peptide with1 non-conservative substitution and 3 conservative substitutionsrelatively to SEQ ID NO:1. The score of the peptide will be16+(3*0.5)=17.5. The corresponding percent similarity will be17.5/20=87.5%.

In a preferred embodiment, the glycosylation pattern of the tandemrepeat is selected from the group consisting of:

(SEQ ID NO. 1) VT^(Tn)SAPDTRPAPGST^(Tn)APPAHG- (SEQ ID NO. 1)VT^(Tn)SAPDTRPAPGS^(Tn)T^(Tn)APPAHG- (SEQ ID NO. 1)VT^(Tn)SAPDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG- (SEQ ID NO. 1)VT^(Tn)S^(Tn)APDTRPAPGS^(Tn)T^(Tn)APPAHG- (SEQ ID NO. 1)VT^(Tn)S^(Tn)APDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG- (SEQ ID NO. 1)VT^(STn)SAPDTRPAPGST^(STn)APPAHG- (SEQ ID NO. 1)VT^(STn)SAPDTRPAPGS^(STn)T^(STn)APPAHG- (SEQ ID NO. 1)VT^(STn)SAPDT^(STn)RPAPGS^(STn)T^(STn)APPAHG- (SEQ ID NO. 1)VT^(STn)S^(STn)APDTRPAGS^(STn)T^(STn)APPAHG- (SEQ ID NO. 1)VT^(STn)S^(STn)APDT^(STn)RPAPGS^(STn)T^(STn)APPAHG-

In an even more preferred embodiment, the glycosylation pattern of thetandem repeat is selected from the group consisting of:

(SEQ ID NO. 1) VT^(Tn)SAPDTRPAPGS^(Tn)T^(Tn)APPAHG-  (SEQ ID NO. 1)VT^(Tn)S^(Tn)APDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG- (SEQ ID NO. 1)VT^(STn)SAPDTRPAPGS^(STn)T^(STn)APPAHG-  (SEQ ID NO. 1)VT^(STn)S^(STn)APDT^(STn)RPAPGS^(STn)T^(STn)APPAHG- 

An advantageous effect has been reported by combining more than onetandem repeat for immunization. Thus, in a preferred embodiment, theimmunogenic glycopeptide comprises more than one tandem repeat, such asmore than 2 tandem repeats, such as more than 3 tandem repeats, such asmore than 4 tandem repeats, such as more than 5 tandem repeats, such asmore than 6 tandem repeats, such as more than 7 tandem repeats, such asmore than 8 tandem repeats such as more than 9 tandem repeats, and suchas more than 10 tandem repeats.

Further, in a preferred embodiment, the immunogenic glycopeptide iscoupled to a suitable carrier selected from the group consisting of:human serum albumin, keyhole limpet hemocyanin (KLH), thyroglobulin,ovalbumin, influenza hemagglutinin, PADRE polypeptide, malariacircumsporozite (CS) protein, hepatitis B surface antigen (HBSAgI9-2s),Heat Shock Protein (HSP) 65, Mycobacterium tuberculosis, cholera toxin,cholera toxin mutants with reduced toxicity, diphtheria toxin, CRM 97protein that is cross-reactive with diphtheria toxin, recombinantStreptococcal C5a peptidase, Streptococcus pyogenes ORF1224,Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF2452,Chlamydia pneumoniae ORF T367, Chlamydia pneumoniae ORF T858, Tetanustoxoid or HIVgp120T1.

Coupling to a carrier is done to increase the potency of theimmunological peptide.

Pharmaceutical Composition

Since the immunogenic glycopeptide can induce a cancer specific immuneresponse, another aspect of the present invention is a pharmaceuticalcomposition comprising the immunogenic glycopeptide.

In a preferred embodiment, the pharmaceutical composition is a cancervaccine for treatment or prevention of breast cancer, ovarian cancer,pancreatic cancer, or lung cancer.

Method of Treating or Preventing Cancer

Another aspect of the invention is a method of treating or preventingcancer comprising administrating the aforementioned pharmaceuticalcomposition comprising the immunogenic glycopeptide. In doing so, acancer specific immune response will be raised.

Antibodies

Other aspects of the present invention are antibodies prepared using theimmunogenic glycopeptide, methods for preparation of said antibodies anduse of said antibodies in therapy and diagnosis.

Thus, another aspect of the present invention is a method for thepreparation of hybridoma cells, which secrete monoclonal antibodiesspecific for the immunogenic glycopeptide characterized in that:

-   -   a suitable mammal is immunized with the immunogenic        glycopeptide,    -   antibody-producing cells of said mammal are fused with cells of        a continuous cell line,    -   the hybrid cells obtained in the fusion are cloned, and    -   cell clones secreting the desired antibodies are selected.

Still another aspect is a monoclonal antibody selected from the groupconsisting of:

-   -   A monoclonal antibody produced by the hybridoma cells prepared        by the method described above    -   A monoclonal antibody prepared by molecular display techniques,        such as mRNA display, ribosome display, phage display and        covalent display against the immunogenic glycopeptide.

Traditionally, monoclonal antibodies have been prepared using hybridomatechnology. However, alternative techniques such as mRNA display,ribosome display, phage display and covalent display are now available.These are all display techniques where a peptide library is selectedagainst the immunogenic glycopeptide. Such techniques can e.g. be usedto identify humanized or fully human antibodies.

In a preferred embodiment, the monoclonal antibody binds MUC1 on cancercells but not MUC1 on a non-malignant counterpart.

In another preferred embodiment, the monoclonal antibody bindspreferentially to MUC1 that is aberrantly glycosylated and expressed oncancer cells.

In still another embodiment, the monoclonal antibody binds to or atleast interacts directly with the O-glycosylated GSTA (SEQ ID NO. 7)motif of the immunogenic glycopeptide. Our data strongly indicate thatantibodies binding to the O-glycosylated GSTA (SEQ ID NO. 7) motif areindeed cancer specific and that cancer specificity may lie in thisinteraction. Not intended to be bound by theory, we believe thatantibodies that bind to or interact with the O-glycosylated GSTA (SEQ IDNO. 7) motif will display cancer specificity. In particular so, if theybind or interact with the O-glycosylated GSTA (SEQ ID NO. 7) motifcarrying an O-glycosylation at the S-residue and the T-residue at thesame time.

In a preferred embodiment, the antibody prepared using the immunogenicglycopeptide is humanized or fully human, such as to decrease theimmunogenicity of the antibody in humans. This is typically desirable ifthe antibody is used as a therapeutic.

However, in some situations a rapid clearance may be desired, whereforealso non-humanized antibodies are of interest as therapeutics. One suchsituation can e.g. be when administering antibodies coupled to toxins orradioisotopes. Such conjugated antibodies should either find theirtarget rapidly or be cleared as they have a general toxic effect. Oneembodiment of the invention is conjugated antibodies.

Another embodiment of the invention is the monoclonal antibody, 5E5,secreted by the hybridoma deposited at the European Collection of CellCultures (ECACC), Centre for Emergency Preparedness and Response of TheHealth Protection Agency, Porton Down, Salisbury SP4 OJG, a depositoryin compliance with the Budapest Treaty of 1977, on Sep. 19, 2006, underaccession number STHM1 06092102.

Another embodiment of the invention is the monoclonal antibody, 2D9,secreted by the hybridoma deposited at ECACC, Centre for EmergencyPreparedness and Response of The Health Protection Agency, Porton Down,Salisbury SP4 OJG, a depository in compliance with the Budapest Treatyof 1977, on Sep. 19, 2006, under accession number: STHM2 06092101.

The aforementioned deposits were made by Mads Agervig Tarp on Sep. 19,2006. The deposit was given the following reference number: Q6847.

Another aspect of the invention is the use of a monoclonal antibody,prepared using the immunogenic glycopeptide, as a medicament.

In a preferred embodiment, the medicament is used for treatment orprevention of cancer.

Still another aspect is the use of a monoclonal antibody, prepared usingthe immunogenic glycopeptide, for the preparation of a medicament forthe treatment or prevention of cancer.

Since monoclonal antibodies prepared using the immunogenic glycopeptidedisplay cancer specificity, a further aspect of the invention is apharmaceutical composition comprising the monoclonal antibody preparedusing the immunogenic glycopeptide.

In a preferred embodiment, the antibody of the pharmaceuticalcomposition is conjugated to a toxin or a radionuclide.

Still another aspect of the invention is a method of determining whetheran individual has cancer or is at risk of developing cancer comprisingthe steps of:

-   -   Providing a sample from the individual    -   Contacting the antibody prepared using the immunogenic        glycopeptide with the sample    -   Removing antibodies not interacting with the sample    -   From the antibodies interacting with the sample, determine        whether the individual has cancer or is at risk of developing        cancer

Still another aspect of the invention is an ex vivo-method of producinga population of autologous antigen presenting cells (APCs), which arecapable of inducing effective immune responses against MUC1, comprisingthe steps of:

-   -   providing autologous APCs from a tumor patient    -   contacting the autologous APCs from the tumor patient with an        effective amount of the immunogenic glycopeptide of the        invention, wherein said contacting is under conditions which        allow endocytosis, processing, and MHC class II presentation of        fragments of said glycopeptide or fusion molecule by said APCs;        and    -   isolating said peptide or fusion molecule fragment-presenting        APCs for the purpose of immunotherapeutic application in the        patient

Still another aspect of the present invention is a method of determiningthe presence of antibodies binding to the immunogenic glycopeptide,comprising the steps of:

-   -   a. Providing a sample comprising human antibodies    -   b. Contacting the sample with a peptide inhibitor and an        O-glycan carbohydrate inhibitor    -   c. Further contacting the sample of step b with the glycopeptide    -   d. Quantifying the amount of antibodies interacting with the        glycopeptide sample

Man has natural antibodies to the Tn, STn and T carbohydrate structuresand these appear to be increased in cancer patients. It is believed thatsuch antibodies will react with the corresponding MUC1 glycopeptidessimilar to the panel of monoclonal anti-Tn, -STn and -T antibodiesanalyzed. In order to identify novel MUC1 glycopeptide antibodies inhuman serum, it is therefore necessary to develop assays that canselectively identify glycopeptide specific antibodies withoutinterference from anti-peptide or anti-carbohydrate antibodies.

While not intended to be bound by theory, it is believed that a peptideinhibitor and an O-glycan carbohydrate inhibitor can be used toneutralize cross-reacting antibodies, without affecting antibodiesspecific for the immunogenic glycopeptide, i.e. antibodies that bind theimmunogenic glycopeptide, but not carbohydrates alone or thenon-glycosylated peptide. Thus, the presence of antibodies specific forthe immunogenic glycopeptide can be detected and even quantified. Sinceit has been demonstrated that these antibodies are cancer specific, themethod can be used for diagnosis and prognosis in relation to cancer.

The sample may be serum, plasma, body fluids such as milk, saliva,mucosal secretions, feces, urine and any antibody preparations hereof.

The peptide inhibitor will typically be a peptide of the same amino acidsequence as the immunogenic glycopeptide, however, without anyglycosylations. In another embodiment, the peptide inhibitor comprisesfully processed branched core 2 O-glycans, as is typically present innormal cells.

The carbohydrate inhibitor will typically be Tn, STn or T. Alsopreferred are polyvalent PAA conjugates of the aforementionedcarbohydrates. Still in another embodiment, the carbohydrate inhibitoris a monosaccharide such as GalNAc, GlcNAc, Gal, Glc and NeuAc. It willbe apparent to the skilled man that other combinations of carbohydrateswill have the same effect.

In a preferred embodiment, the method further comprises a step ofremoving antibodies that interact with the peptide inhibitor and/or withthe O-glycan carbohydrate inhibitor.

In still another embodiment, the peptide inhibitor and the O-glycaninhibitor has been immobilized on a solid support, which is used toremove antibodies that interact with the peptide inhibitor and/or withthe O-glycan carbohydrate inhibitor.

In still another embodiment, the antibodies binding to the immunogenicglycopeptide, binds to the O-glycosylated GSTA (SEQ ID NO. 7) motif ofthe immunogenic glycopeptide. As is apparent from the presentspecification, such antibodies are cancer specific and their presencemay indicate that the individual has cancer.

Thus, in another preferred embodiment

-   -   a. the sample is provided from an individual that is suspected        of having cancer    -   b. the determined amount of antibodies that interact with the        glycopeptide is compared to a standard amount, said standard        amount being determined from a control group    -   c. a determined amount of antibodies above the standard amount        is indicative of cancer in the individual    -   d. a determined amount of antibodies below the standard amount        is indicative of the individual not having cancer

It is noted that the above described method of determining the presenceof antibodies binding to the immunogenic glycopeptide is not necessarilylimited to the immunogenic glycopeptide of the present invention. Themethod should be applicable for detection of antibodies specificallybinding (not binding to peptide or carbohydrate alone) to otherglycopeptides as well.

FIGURE LEGENDS

FIG. 1. Chemoenzymatic synthesis of multimeric Tn and STn MUC1glycopeptides:

Synthetic 60-mer MUC 1 tandem repeat peptides(VTSAPDTRPAPGSTAPPAHG)_(n=3) (SEQ ID NO. 1) were glycosylated usingsite-selective recombinant polypeptide GalNAc-transferases (GalNAc-T2,-T4 and -T11). The sites of GalNAc attachments in MUC1 tandem repeatsequences were strictly controlled as indicated by MALDI-TOF massspectrometry analysis of MUC1 60-mer tandem repeat peptides glycosylatedin vitro with recombinant GalNAc-transferases. GalNAc-T11 was used toadd 2 GalNAc residues per tandem repeat, GalNAc-T2 to add 3 residues,and sequential use of GalNAc-T2 and -T4 to add all 5 residues. Sites ofattachments were confirmed by mass spectrometry as previously described.Glycosylation with GalNAc-T4 to achieve five GalNAc residues per repeatonly allowed 14 in total due to the design of the peptide with theNH₂-terminal being too truncated. Further glycosylation of GalNAcresidues with sialic acid to form STn was achieved with recombinantmurine ST6GalNAc-I. Evaluation of number of sialic acid residuesattached by MALDI-TOF may be underestimated due to the labile nature ofthis sugar linkage. The sialylation is considered complete as evaluatedby immunoreactivity pattern with anti-STn (positive) and anti-Tn(negative) monoclonal antibodies. The core 1 T structure was producedusing a recombinant β3Gal-transferase. Glycopeptides formed are depictedon top of each MALDI-TOF profile. The mass scale of spectra shown are5,000-10,000 counts.

FIG. 2. MUC1 glycopeptides with complete O-glycan attachment are mostimmunogenic and Tn and STn glycopeptides elicit strong antibodyresponses in MUC1 transgenic mice.

(a) ELISA assay of serum from one representative (of four) wild-typeBalb/c mouse immunized with complete Tn glycosylated MUC1 (MUC1₆₀Tn₁₅).Designations are as follows: ▪=MUC1₆₀Tn₁₅; □=MUC1₆₀Tn₉; ◯=MUC1₆₀STn₁₅;●=OSM (STn); ▴=MUC1₆₀Tn₆; Δ=MUC1₆₀; ▴=AOSM (Tn). Additional peptidestested which gave no reactivity include unglycosylated MUC2, Tn MUC2,and Tn MUC4. (b) ELISA assay of serum from one representative (of four)wild-type mouse immunized with the complete STn glycosylated MUC1glycopeptide (MUC1₆₀Tn₁₅). The highest antibody titers were found withthe MUC1 glycoform used as immunogen, but considerable reactivity withthe other MUC1 glycoforms were found as well. Low reactivity withunglycosylated MUC1 was found in particularly in the Tn immunized mice.Very low levels of anti-Tn and STn hapten antibodies were detected usingthe mucin OSM (ovine submaxillary mucin with mainly STn glycoform) andAOSM (asialo-mucin with Tn glycoform) as antigens. No reactivity withnon-MUC1 peptides or glycopeptides with Tn-glycosylation were found. (c)ELISA assay of serum from one (of four) MUC1.Tg mice immunized withcomplete Tn glycosylated MUC1 (MUC1₆₀Tn₁₅). (d) ELISA assay of serumfrom one (of four) MUC1.Tg mice immunized with the complete STnglycosylated MUC1 glycopeptide (MUC1₆₀STn₁₅). The highest antibodytiters were found with the MUC1 glycoform used as immunogen, butconsiderable reactivity with the other MUC1 glycoforms was found aswell. No reactivity was detected with unglycosylated MUC1 as well as themucins OSM (STn) and AOSM (Tn) and non-MUC1 Tn glycopeptides.

FIG. 3. Characterization of a monoclonal antibody 5E5 that mimics theimmune response elicited in wild type and MUC1.Tg mice immunized with TnMUC 1.

(a) ELISA assay with monoclonal antibody 5E5 shows strong reactivitywith all glycoforms of the MUC1 tandem repeat sequence, but noreactivity with the unglycosylated MUC1 peptide. Weak reactivity wasalso observed with AOSM, but no reactivity was detected with other Tnglycopeptides. Designations as in FIG. 2. Negative control peptidesinclude unglycosylated MUC2, Tn MUC2, Tn MUC4Tn₁ and Tn MUC4Tn₃. (b)Immunofluorescence staining with Mab 5E5 (top row) showing reactivitywith CHO ldlD cells expressing the Tn MUC1 glycoform, no reactivity withcells expressing unglycosylated MUC1, ST MUC1 or T MUC1 (afterpretreatment with neuraminidase) glycoforms as well as wild type CHOldlD cells. Control antibodies to MUC1 (HMFG2), Tn (5F4) and T (HH8)were included to confirm the expression of MUC1 and the respectiveglycoforms Tn, T, and ST as indicated (c) SDS-PAGE Western blot analysisof culture medium of CHO ldlD cells secreting different MUC1-glycoforms.Monoclonal antibody 5E5 exhibits strict specificity for the secreted TnMUC1 glycoform, while HMFG2 reacts with all glycoforms as well asunglycosylated MUC1. (d) Immunohistochemical staining of breast tissueswith monoclonal antibody 5E5. Primary breast infiltrating ductalcarcinoma grade II stained with 5E5. Note that surrounding normal tissueis negative (A). Ductal carcinoma in situ stained with 5E5 (B). Grade IIductal carcinoma showing areas of DCIS. Both infiltrating and DCIS arestaining with 5E5 (C). Primary breast infiltrating ductal carcinomagrade III stained with 5E5 (D).

FIG. 4. MUC1.Tg mice immunized with MUC1 Tn and STn glycopeptidesproduce MUC1 glycopeptide specific responses restricted tocancer-associated MUC1 glycoforms.

(a) Sera from MUC1.Tg mice immunized with MUC1 Tn or STn glycopeptidesreacted with Tn MUC1 but not unglycosylated or T/ST glycoforms of MUC1expressed in CHO ldlD cells. Sera from Tg mice immunized withunglycosylated MUC1 reacts preferentially, but weakly with CHO ldlDcells expressing unglycosylated MUC1. (b) Sera from Tg mice immunizedwith MUC1 glycopeptides recognize MUC1 expressed by cancer cells.Immunohistochemical staining of a primary breast carcinoma expressingboth STn (B) and MUC1 (A) (determined by monoclonal antibodies HB-STnand HMFG2) with serum from one Tg mouse immunized with MUC160STn15 (C).

FIG. 5. Glycopeptides used for characterization of MAb specificities.

Biotinylated 60-mer glycopeptides: (VTSAPDTRPAPGSTAPPAHG)n=3 (SEQ ID NO.1)

Prefix numbers indicate number of O-glycans in peptides. Tn:GalNAcα1-O-Ser/Thr; STn: NeuAcα2-6GalNAcα1-O-Ser/Thr; T:Galβ1-3GalNAcα1-O-Ser/Thr; ST: NeuAcα2-3Galβ1-3GalNAcα1-O-Ser/Thr; core3: GlcNAcβ1-3GalNAcα1-O-Ser/Thr.

Biotinylated 25-mer valine-substituted glycopeptides: TAP25V9:

(TAPPAHGVTSAPDTRPAPGSVAPPA) (SEQ ID NO. 11) Valine-substituted inposition 9; TAP25V21: Valine-substituted in position 21; 2Tn-TAP25V9 and2Tn-TAP25V21 are glycosylated with Tn (GalNAcα-O-Ser/Thr) in theindicated positions.

21-mer: (AHGVTSAPDTRPAPGSTAPPA) (SEQ ID NO. 12) Synthetic glycopeptideswith a single Tn (GalNAcα1-O-Ser/Thr) or T (Galβ1-3GalNAcα1-O-Ser/Thr)glycan in the indicated positions.

FIG. 6. Specificity analysis of MAbs 2D9 and 5E5 by ELISA.

Panels A and D: Reactivity of MAbs 2D9 and 5E5 with biotinylated 60-merglycopeptides by capture ELISA (FIG. 1). Strong reactivity is seen forboth MAbs with high-density Tn and STn glycoforms.

Panels B and E: Reactivity of MAbs 2D9 and 5E5 with biotinylatedvaline-substituted 25-mer glycopeptides by capture ELISA. Strongreactivity is seen for both MAbs with the peptide Tn-glycosylated at Thrin the GSTA region. □ indicates Tn-glycosylation.

Panels C and F: Reactivity of MAbs 2D9 and 5E5 with 21-mer glycopeptidesglycosylated with a single Tn or T glycan by direct binding ELISA.Strong reactivity is seen for both MAbs with the peptide Tn-glycosylatedat Thr in the GSTA region. □ indicates Tn-glycosylation.

Controls for 25- and 21-mer peptides are shown with MAb 5E10 in FIG. 5,panels B and C.

FIG. 7. Specificity analysis of serum from MUC1 transgenic miceimmunized with 15Tn-MUC1 60-mer glycopeptide.

Panel A: Reactivity with 60-mer glycopeptides by direct binding ELISA.Strong reactivity is seen with glycopeptides with three or five Tnglycans per tandem repeat. Lower reactivity is seen with the fullySTn-glycosylated peptide. No reactivity is seen with the unglycosylatedpeptide.

Panel B: Reactivity with biotinylated valine-substituted 25-merglycopeptides by capture ELISA. Strong reactivity is seen with thepeptide Tn-glycosylated at Thr in the GSTA region. □ indicatesTn-glycosylation.

FIG. 8. Specificity analysis of MAbs 1B9, BW835 and MY.1E12 by ELISA.

Panel A: Reactivity of MAb 1B9 with biotinylated 60-mer glycopeptides.Reactivity is also seen with core 3 and ST glycans (see text fordetails).

Panel B: Reactivity of MAb 1B9 with biotinylated valine-substituted25-mer glycopeptides. Reactivity is seen with glycopeptides with Tglycans at Thr in the GSTA region and Thr in the VTSA (SEQ ID NO. 6)region.

Panel C: Reactivity of MAb 1B9 with CHO ldlD cells grown in Gal/GalNAc.1B9 reacts with approximately 2% of the cells expressing ST-MUC1 (noneuraminidase treatment), whereas it reacts with approximately 20% ofcells presenting T-MUC1 (neuraminidase treated).

Panel D: Reactivity of MAb BW835 with biotinylated 60-mer glycopeptides.Published epitope listed in parenthesis where ∘ indicatesT-glycosylation. Strong reactivity is seen with the glycopeptides withfive T or core 3 glycans per tandem repeat. Lower reactivity is observedwith five ST glycans per tandem repeat.

Panel E: Reactivity of MAb MY.1E12 with biotinylated 60-merglycopeptides. Published epitope listed in parenthesis where * indicatesST-glycosylation. Strong reactivity is seen with the glycopeptide withfive ST glycans per tandem repeat. Lower reactivity is observed withthree ST glycans per tandem repeat.

FIG. 9. Specificity analysis of MAbs 5E10 and SM3 by ELISA.

Panel A: Reactivity of MAb 5E10 with biotinylated 60-mer glycopeptides.Reactivity is seen with all tested peptides except when fullyglycosylated with the STn glycan. Preference is seen for unglycosylatedand Tn glycoforms, followed by T and ST glycoforms. Lowest reactivity isseen with the glycopeptide with three STn glycans per tandem repeat.

Panel B: Reactivity of MAb 5E10 with biotinylated valine-substituted25-mer glycopeptides. Strong reactivity is seen with all peptidesindependent on Tn-glycosylation.

Panel C: Reactivity of MAb 5E10 with 21-mer glycopeptides glycosylatedwith a single Tn or T glycan. Relatively strong reactivity is seen withall glycopeptides with exception of the peptide with a T glycan at Thrin the PDTR (SEQ ID NO. 4) region.

Panel D: Reactivity of MAb SM3 with biotinylated 60-mer glycopeptides.Strongest reactivity is seen with unglycosylated peptide or peptideswith five O-glycans per tandem repeat. The glycoforms of preference areTn, STn, core 3, T, and ST in the mentioned order.

Panel E: Reactivity of MAb SM3 with biotinylated valine-substituted25-mer glycopeptides. Weak reactivity is seen with the unglycosylatedpeptides and where the Thr in the GSTA (SEQ ID NO. 7) region isTn-glycosylated. No reactivity is seen with the glycopeptideglycosylated in the Thr in the VTSA (SEQ ID NO. 6) region.

Panel F: Reactivity of MAb SM3 with 21-mer glycopeptides glycosylatedwith a single Tn or T glycan. Strongest reactivity is seen withglycopeptides with T- or Tn-glycosylation at Thr in the PDTR (SEQ ID NO.4) region. Low reactivity is seen with the remaining T-glycosylated andsome of the Tn-glycosylated glycopeptides.

EXAMPLES Example 1 Materials and Methods Chemoenzymatic Synthesis ofMultimeric Tn and STn MUC1 Glycopeptides

MUC1 60-mer (VTSAPDTRPAPGSTAPPAHG)n=3 (SEQ ID NO. 1) peptide wassynthesized as originally reported by Fontenot. Control peptides usedwere derived from tandem repeat of MUC2 (PTTTPISTTTMVTPTPTPTC) (SEQ IDNO. 8) and MUC4 (CPLPVTDTSSASTGHATPLPV) (SEQ ID NO. 9). Peptides wereglycosylated in vitro using purified recombinant humanglycosyltransferases polypeptide GalNAc-T2, GalNAc-T4, and GalNAc-T11.The GalNAc substituted peptides were subsequently sialylated usingpurified recombinant mouse ST6GalNAc-I. GalNAc glycosylation of thepeptides was performed in a reaction mixture (1 mg peptide/ml)containing 25 mM cacodylate buffer (pH 7.4), 10 mM MnCl2, 0.25% TritonX-100, and 2 mM UDP-GalNAc. Glycosylation of 1 mg 60-mer peptide with 2GalNAc per TR (MUC160Tn6) was obtained using GalNAc-T11. Incorporationof 3 GalNAc per TR (MUC160Tn9) was obtained using GalNAc-T2.Substitution of all five putative O-glycosylation sites in the MUC1 TR(MUC160Tn15) was performed using MUC160Tn9 as substrate in a reactionwith GalNAc-T4. Sialylation was performed in a reaction mixture (1 mgpeptide/ml) containing 20 mM Bis-Tris buffer (pH 6.5), 20 mM EDTA, 1 mMdithiothreitol and 2 mM CMP-NANA (Sigma). Glycosylation was monitoredusing nano-scale reversed-phase columns (Poros R3, PerSeptive Biosystem)and MALDI-TOF mass spectrometry. The glycopeptides were purified by HPLCon a Zorbax 300SB-C3 column (9.4 mm×25 cm) in an 1100 Hewlett Packardsystem using 0.1% TFA and a gradient of 0-80% acetonitrile.Quantification and estimation of yields of glycosylation reactions wereperformed by comparison of HPLC peaks by uv 210 absorbance using 10 μgweighed peptide as standard. GalNAc-glycosylation of peptides generallyyielded 80-90% recovery, while the sialylation step was more variablewith yields from 60-80%. Purified glycopeptides were characterized byMALDI-TOF mass spectrometry on a Voyager DE or Voyager DE Pro MALDItime-of-flight mass spectrometer (PerSeptive Biosystems Inc.,Framingham, Mass.) equipped with delayed extraction. The MALDI matrixwas 2,5-Dihydroxybenzoic acid 10 g/L (Aldrich, Milwaukee, Wis.)dissolved in 2:1 mixture of 0.1% trifluoroacetic acid in 30% aqueousacetonitrile. Samples dissolved in 0.1% trifluoroacetic acid to aconcentration of approximately 1 pmol/μl were prepared for analysis byplacing 1 μl of sample solution on a probe tip followed by 1 μl ofmatrix. All mass spectra were obtained in the linear mode. Dataprocessing was carried out using GRAMS/386 software.

Immunization Protocol

Glycopeptides were coupled to keyhole limpet hemocyanin (KLH) (Pierce,Rockford, Ill.) using glutaraldehyde. Efficiency of conjugation wasassessed by analyzing the reaction by size exclusion chromatography on aPD-10 column using anti-MUC1 ELISA of fractions. Essentially allreactivity was found was found with the excluded fraction andinsignificant reactivity in the included fractions expected to containpeptides. Further evaluation included comparative titration analysis ofthe KLH conjugate with the corresponding glycopeptide in ELISA. Bothanalyses indicated that the conjugation was near complete, which shouldresult in a KLH to glycopeptide ratio of 1:300. MUC1 transgenic mice(MUC1.Tg) homozygous for the transgene expression were originallydeveloped on an H2-k background. Subsequently, these mice have beenbackcrossed onto a Balb/c strain for 15 generations to give a pureBalb/c (H2-d) background (Graham and Taylor-Papadimitriou, unpublisheddata). Female Balb/c wild type and MUC1.Tg mice were injectedsubcutaneously with 10 or 15 μg of (glyco)peptide in a total volume of200 μl (1:1 mix with Freunds adjuvant, Sigma). Mice received fourimmunizations 14 days apart, and blood samples were obtained by tail oreye bleeding 1 week following the third and fourth immunization.

Generation of Mouse Monoclonal Anti-Tn-MUC1 Antibody 5E5.

A monoclonal antibody was produced as described previously from a wildtype Balb/c mouse immunized with the fully GalNAc-glycosylated 60-merMUC1 glycopeptide coupled to KLH. Screening was based on glycopeptideELISA assays followed by immunocytology with breast cancer cell lines(MCF7, T47D, MTSV1-7) and immunohistology with breast cancer tissues.Selection was based on reactivity pattern similar to total sera of thesame mouse.

ELISA Assays

Enzyme-linked immunosorbent assays (ELISA) were performed using 96-wellMaxiSorp plates (Nunc, Denmark). Plates were coated overnight at 4° C.with 1 μg/ml of glycopeptides in bicarbonate-carbonate buffer (pH 9.6),blocked with 5% BSA in PBS, followed by incubation with sera (diluted inPBS) or monoclonal antibodies for 2 hours at room temperature. Boundantibodies were detected with peroxidase-conjugated rabbit anti-mouseimmunoglobulins (Dako, Denmark) or isotype specific antibodiesperoxidase-conjugated goat anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3(Southern Biotechnology Associates, USA). Plates were developed withO-phenylenediamine tablets (Dako, Denmark) and read at 492 nm. Controlantibodies included anti-MUC1 antibodies HMFG2 and SM3 andanti-carbohydrate antibodies 5F4 (Tn) and 3F1 (STn). Control seraincluded mice immunized with MUC4 mucin peptide linked to KLH.

Cell Lines

The human mammary cell lines MCF7, MTSV1-7, and T47D, and the murinepancreatic carcinoma cell line Panc02 were cultured as previouslydescribed. CHO ldlD cells were stably transfected with full coding MUC1containing 32 tandem repeats and grown with or without addition ofGal/GalNAc as indicated. Confluent cultures of CHO ldlD cells in 6 wellplates (Nunc, Denmark) were grown in HAM'S F12 with 10% FCS withoutGalNAc and Gal, in presence of 1 mM GalNAc, or in the presence of 1 mMGalNAc and 0.1 mM Gal (Sigma Aldrich). The medium was harvested after 48hours of growth and used for immunoassays. Cells were trypsinized,washed and airdried on coverslides for immunocytology.

SDS-PAGE Western Blot

SDS-PAGE western blot analysis was performed according to manufacturersinstructions (4-12% gradient gel, Biowhittaker Molecular Applications).Membranes were blocked in 15% skimmed milk powder (Merck Eurolab),incubated with MAbs 5E5 and HMFG2 overnight at 4° C., followed byincubation with biotinylated goat anti-mouse IgG1 (0.5 μg/ml)(SouthernBiotechnology Inc) for 1 hour at room temperature. Membranes wereincubated with avidin horseradish peroxidase conjugate (0.36 μg/ml)(Dako) for 30 min at room temperature, followed by 50 mM Tris-HCl buffer(pH 7.6) containing 0.04% 4-chloro-1-naphthol (Sigma) and 0.025% H₂O₂.

Immunocytochemistry

Cell lines were fixed for 10 min in ice cold acetone or inmethanol:acetone. Fixed cells were incubated overnight at 5° C. withmouse sera (1:200/1:400/1:800) or monoclonal antibodies, followed byincubation for 45 min at room temperature with FITC-conjugated rabbitanti-mouse immunoglobulins (Dako, Denmark). Slides were mounted inglycerol containing p-phenylenediamine and examined in a Zeissfluorescence microscope.

Immunohistochemistry

Frozen tissue samples were fixed for 10 min in cold methanol/acetone(50:50). Formalin fixed, paraffin wax embedded tissues of breastcarcinoma were obtained from files of Institute of Molecular Pathologyand Immunology of the University of Porto, Portugal. All cases wereconventionally classified by histological type. Theavidin-biotin-peroxidase complex method was used for immunostaining.Paraffin sections were dewaxed, rehydrated, and treated with 0.5% H₂O₂in methanol for 30 min. Section were rinsed in TBS and incubated for 20min with rabbit nonimmune serum. Sections were rinsed and incubatedovernight at 5° C. with primary antibody. Sections were rinsed andincubated with biotin-labeled rabbit anti-mouse serum (Dako, Denmark)diluted 1:200 in TBS doe 30 min, rinsed with TBS, and incubated for 1 hwith avidin-biotin-peroxidase complex (Dako, Denmark). Sections wererinsed with TBS and developed with 0.05% 3,3′-diaminobenzidinetetrahydrochloride freshly prepared in 0.05 M TBS containing 0.1% H₂O₂.Sections were stained with hematoxylin, dehydrated and mounted.

Results Chemoenzymatic Synthesis of Multimeric Tn and STn MUC1Glycopeptides.

Synthetic 60-mer MUC1 tandem repeat peptides were glycosylated usingsite-selective recombinant polypeptide GalNAc-transferases (GalNAc-T2,-T4 and -T11). The sites of GalNAc attachments in MUC1 tandem repeatsequences were strictly controlled as indicated by MALDI-TOF massspectrometry analysis of MUC1 60-mer tandem repeat peptides glycosylatedin vitro with recombinant GalNAc-transferases. GalNAc-T11 was used toadd 2 GalNAc residues per tandem repeat, GalNAc-T2 to add 3 residues,and sequential use of GalNAc-T2 and -T4 to add all 5 residues (FIG. 1).Sites of attachments were confirmed by mass spectrometry as previouslydescribed. Glycosylation with GalNAc-T4 to achieve five GalNAc residuesper repeat only allowed 14 in total due to the design of the peptidewith the NH₂-terminal being too truncated. Further glycosylation ofGalNAc residues with sialic acid to form STn was achieved withrecombinant murine ST6GalNAc-I. Evaluation of number of sialic acidresidues attached by MALDI-TOF may be underestimated due to the labilenature of this sugar linkage. The sialylation is considered complete asevaluated by immunorecativity pattern with anti-STn (positive) andanti-Tn (negative) monoclonal antibodies. The core 1 T structure wasproduced using a recombinant β3Gal-transferase. Glycopeptides formed aredepicted on top of each MALDI-TOF profile in FIG. 1.

MUC1 Glycopeptides with Complete O-Glycan Attachment are MostImmunogenic and Tn and STn Glycopeptides Elicit Strong AntibodyResponses in MUC1 Transgenic Mice.

In initial studies MUC1 Tn glycoforms with 2, 3 and 5 O-glycans perrepeat were tested as immunogens, and the glycopeptide with 3 and 5O-glycans yielded the strongest immune response to the respectiveimmunogens by ELISA and more importantly induced antibodies reactivewith MUC1 expressing cancer cells (not shown). For the further studiesMUC1 with complete O-glycan occupancy was chosen, and as shown in FIG. 2sera from wild-type Balb/c mice (FIG. 2 ac) and MUC1.Tg mice (FIG. 2 bd)immunized with either the complete Tn glycosylated MUC1 (MUC1₆₀Tn₁₅) orthe complete STn glycosylated MUC1 glycopeptide (MUC1₆₀STn₁₅) yieldedhigh antibody titers in both mice. The highest antibody titers werefound with the MUC1 glycoform used as immunogen, but considerablereactivity with the other MUC1 glycoforms were found as well. Lowreactivity with unglycosylated MUC1 was found particularly in the Tnimmunized mice. Very low levels of anti-Tn and STn hapten antibodieswere detected using the mucin OSM (ovine submaxillary mucin with mainlySTn glycoform) and A-OSM (asialo-mucin with Tn glycoform) as antigens.No reactivity with non-MUC1 peptides or glycopeptides withTn-glycosylation were found.

Characterization of a Monoclonal Antibody 5E5 That Mimics the ImmuneResponse Elicited in Wild Type and MUC1.Tg Mice Immunized with Tn MUC1.

In order to further characterize and define the specificity of theimmune response to the glycopeptides, we isolated a monoclonal antibody(designated 5E5) from a mouse immunized with the complete Tnglycosylated MUC1 glycopeptide, which essentially mirrored thespecificity of the polyclonal response found (FIG. 3a ). The antibody5E5 reacted with all Tn and STn glycoforms of the MUC1 tandem repeat andshowed no reactivity with unglycosylated MUC1 peptides and only veryweak reactivity with the Tn hapten presented on non-MUC1 peptidebackbone. In order to evaluate the range of O-glycan structures involvedin the specificity we took advantage of the CHO ldlD cell system. CHOldlD cells lack the UDP-Gal/GalNAc epimerase and are deficient in GalNAcO-glycosylation and galactosylation in the absence of exogenous additionof GalNAc and Gal, respectively. CHO ldlD cells stably transfected withthe full coding human MUC1 gene (CHO ldlD/MUC1) were grown in thepresence of GalNAc, in the presence of Gal and GalNAc or in the absenceof both, yielding cells expressing Tn, ST, or unglycosylated MUC1glycoforms, respectively. As shown in FIG. 3b the CHO ldlD MUC1 cellsexpress MUC1 as detected by the general anti-MUC1 antibody HMFG2regardless of addition of sugars to the growth medium. Cells grown inGalNAc alone express as expected only Tn antigen and not T or ST, whilecells grown in Gal and GalNAc as expected only express ST.Interestingly, cells grown in GalNAc alone do not express the STnstructure, which indicate that the CHO ldlD cells do not expresssignificant amounts ST6GalNAc-I. The staining of the anti-carbohydrateantibodies was highly dependent on expression of MUC1 sincenon-transfected CHO ldlD cells only showed very weak reactivity (notshown). Further confirmation of the MUC1 glycoforms produced by CHO ldlDcells have been achieved by mass spectrometric analysis of a secretedMUC1-IgG chimeric construct grown with or without sugars (results to bepublished elsewhere). 5E5 specifically reacted with the Tn glycoform ofrecombinant MUC1 expressed in the CHO ldlD cells and did not react withunglycosylated or further glycosylated T and ST MUC1 glycoforms (FIG. 3bc). 5E5 defined a cancer-associated glycoform of MUC1 stronglyexpressed in most breast cancers (Table I, FIG. 3d ). 5E5 stained allductal carcinomas (n=18) and 2 lobular carcinomas. The percentage ofpositive cells varied among less than 25% to more than 75%. 6 cases ofbenign lesions were stained, of these only 2 (1 fibrosis and 1fibroadenoma) showed positive staining with 5E5 and in these cases lessthan 25% of the cells stained. This staining pattern closely followedthat of monoclonal antibody HMFG2 in cancer, but 5E5 was more restrictedin normal breast and benign lesions. This further indicates that Tn andSTn MUC1 tandem repeat glycopeptides represent prime vaccine candidates.

TABLE I Immunochemical staining of human breast tissue with anti-MUC1monoclonal antibodies 5E5 HMFG2 Proportion Proportion of tumor of tumorTissue cells cells sample Pathology Grade Node stained Intensity stainedIntensity 1014 (B) 1 Normal − − + + 1020 (A2) Normal − − − − 1073 (A)Normal − − − − 1168 (B) Normal − − + ++ 1196 (A) Normal − − + ++ 1076(B) Normal − − − −  950 (B) Normal − − − −  91 (K) Duct − − + ++hyperplasia  585 (A) Duct − − − − hyperplasia  364 (B) Fibrocystic, − −  <25% ++ duct hyperplasia  60 (B) Fibrocystic, − −   <25% + ducthyperplasia  35 (B) Fibrosis   <25% +++   <25% ++ 1309 (B) Fibroadenoma  <25% +++   <25% +++  268H Lobular − 50-75% ++ 50-75% +++ carcinoma  83Lobular + 25-50% +++ ND ND carcinoma  508 K III Ductal II − 50-75% +++50-75% +++ carcinoma  314 G Ductal II − 25-50% +++ 25-50% +++ carcinoma 558 F Ductal II − 50-75% +++ 50-75% +++ carcinoma  658 M Ductal II +50-75% +++   <25% + carcinoma  58 DI Ductal III − 25-50% +++ 25-50% +++carcinoma  390 L Ductal III −   <25% +++   <25% +++ carcinoma  726 H IIIDuctal III −   >75% +++   >75% +++ carcinoma  393 A Ductal III +   <25%+++ 25-50% +++ carcinoma  418 J II Ductal III + 50-75% +++ 50-75% +++carcinoma  341 Ductal III + 50-75% +++ ND ND carcinoma  456 Ductal III +  >75% +++ ND ND carcinoma  57 Ductal I − 50-75% +++ ND ND carcinoma 182 Ductal I − 25-50% +++ ND ND carcinoma  313 E Ductal I + 25-50% +++ND ND carcinoma  579 Ductal I + 25-50% +++ ND ND carcinoma 1185 DuctalI +   <25% +++ ND ND carcinoma  207 Ductal I + 50-75% +++ ND NDcarcinoma  899 Ductal I + − − ND ND carcinoma Intensity: −: no staining;+: weakly positive; ++: moderately positive; +++: strongly positive ND:not determinedMUC1.Tg Mice Immunized with Tn and STn MUC1 Glycopeptides Produce MUC1Glycopeptide Specific Responses Restricted to Cancer-Associated MUC1Glycoforms.

MUC1 tandem repeat peptide vaccines have generally been ineffective ininducing humoral responses to the cancer associated MUC1, when the mucinis expressed as a self antigen, presumably due to tolerance. However, asshown in FIG. 2 both the Tn and STn 60-mer MUC1 glycopeptides inducedstrong antibody responses to the glycopeptides in MUC1 transgenic mice.The specificities of the antibody responses were essentially identicalto those found in wild type mice. The Ig subclass distribution wasprimarily of IgG1, but responses to STn 60-mer MUC1 included IgG2A andIgG2B subclasses indicating significant class switching (not shown). Theelicited antibodies reacted with recombinant Tn MUC1 expressed in CHOldlD cells (FIG. 4a ) similar to wild type sera (not shown) and themonoclonal antibody 5E5 (FIG. 3b ). Furthermore, sera from miceimmunized with Tn MUC1 glycopeptides reacted strongly with the humanbreast cancer cell line T47D, which mainly carry Tn but also some T andST O-glycans. Sera raised against MUC1₆₀Tn₁₅ showed strong staining ofT47D cells. Sera from mice immunized with MUC1 60-mer carrying 2 or 3 Tnper tandem repeat sequence showed intermediate levels of reactivity withthe tumor cell line. Sera from MUC1.Tg mice immunized with MUC1₆₀Tn₁₅showed intermediate staining of T47D. Another breast cancer cell line,MCF7, has been shown to express MUC1 with O-glycans partially based oncore 2 structures and thus has a glycosylation pattern that more closelyresembles the pattern found in normal epithelial cells. Sera from miceimmunized with MUC1₆₀Tn₁₅ showed lower reactivity with MCF7 than withT47D cells. MCF7 was not stained by sera raised against MUC1₆₀STn₁₅. Allsera demonstrated very low reactivity with the non-tumorigenicepithelial cell line MTSV1-7, which expresses high levels of C2GnT1 andproduces MUC1 carrying core 2 based O-glycans. Finally, anti-sera fromthe MUC1 transgenic mice immunized with MUC1₆₀STn₁₅ reacted with primarybreast carcinomas expressing MUC1 and STn (FIG. 4b ).

Example 2 Materials and Methods Chemoenzymatic Synthesis ofGlycopeptides

A MUC1 60-mer peptide (VTSAPDTRPAPGSTAPPAHG)_(n=3) (SEQ ID NO. 1)representing three tandem repeats was synthesized (by Cancer ResearchUK) as originally reported. For immunization of mice, the peptide wascompletely Tn-glycosylated in vitro by concerted action of GalNAc-T2 and-T4 (see below). For capture ELISA, an NH₂-terminal biotinylated variantof the 60-mer peptide was in vitro glycosylated to form 11 differentglycoforms (FIG. 5). Furthermore, as control peptide a MUC2 33-merpeptide (PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC) (SEQ ID NO. 14)corresponding to 1.4 tandem repeat (kindly provided by Dr. P. O.Livingston) was Tn-glycosylated by GalNAc-T2 with an occupancy ofapproximately 12 out of 20 potential acceptor sites. Twovaline-substituted NH₂-terminal biotinylated 25-mer MUC1 peptides,TAP25V9 (T¹APPAHGVV⁹SAPDTRPAPGST²¹APPA) (SEQ ID NO. 10) and TAP25V21(T¹APPAHGVT⁹SAPDTRPAPGSV²¹APPA), (SEQ ID NO. 11) were synthesized andtheir glycosylation products with different polypeptideGalNAc-transferases characterized as previously described. Thesepeptides were enzymatically in vitro glycosylated at Thr¹ and Thr²¹ orThr¹ and Thr⁹, respectively, by using GalNAc-T11 (FIG. 5). Furthermore,eight different 21-mer MUC1 glycopeptides with either a single Tn-glycan(Tn-A1-Tn-A4) or a single T-glycan (T-Al-T-A4) based on the sequenceAHGVTSAPDTRPAPGSTAPPA (SEQ ID NO. 12) were chemically synthesized (FIG.5).

Conjugation of MUC1 60-Mer Glycopeptide for Immunization

60-mer MUC1 peptide carrying 15 GalNAc residues was conjugated toImject® Mariculture Keyhole Limpet Hemocyanin (mcKLH) (PierceBiotechnology, Inc., Rockford, Ill.) using glutaraldehyde in a molarratio of glycopeptide:mcKLH 300:1. Excess glutaraldehyde was removed onPD-10 desalting columns (Amersham Biosciences, Uppsala, Sweden) elutingin PBS. Fractions were pooled based on OD readings at 280 nm and 210 nm.The fractions corresponding to the elution time of the unconjugatedpeptides did not contain peptide according to OD reading at 210 nm.Furthermore, in ELISA, the rate of conjugation was estimated to benearly complete by comparing reactivity of the conjugates and thecorresponding unconjugated glycopeptides with monoclonal antibodiesdirected to the glycopeptide or the glycan alone.

Production of Recombinant MUC1 in CHO ldlD Cells

CHO ldlD cells stably transfected with a soluble MUC1-murine IgG2afusion construct containing 16 tandem repeats were cultured in Iscove'smodified Dulbecco's medium with 10% FCS and 600 μg/ml G418. Exploitingthe deficiency of UDP-Gal/UDP-GalNAc 4-epimerase in these cells,culturing with 1 mM GalNAc yielded cells expressing soluble Tn-MUC1,whereas culturing with 1 mM GalNAc and 0.1 mM Gal yielded cellsexpressing soluble ST-MUC1. Glycoproteins (6xHis-tagged) were purifiedon Ni-NTA agarose (Qiagen, Hilden, Germany). Purified ST-MUC1 wastreated with neuraminidase (0.2 U/ml in 50 mM sodium acetate buffer, pH5.5) to render T-MUC1 followed by re-purification on Ni-NTA agarose forremoval of neuraminidase.

Generation of MAb 2D9

Similarly to MAb 5E5, female Balb/c mice were immunized with 15Tn-MUC160-mer glycopeptide conjugated to KLH. Tail bleeds were collected sevendays after the third immunization and sera tested by ELISA with theTn-MUC2 glycopeptide serving as negative control, or byimmunocytochemistry with CHO ldlD MUC1F cells expressing Tn-MUC1,ST-MUC1 (T-MUC1 after neuraminidase treatment) or unglycosylated MUC1,T47D (human ductal breast carcinoma), MCF7 (human breast carcinoma), andMTSV1-7 (human breast). Three days after the fourth immunization, spleencells from one mouse were fused with NS1 myeloma cells. Hybridomasspecific to the antigens of interest were cloned by limiting dilution atleast three times.

Other Monoclonal Antibodies

Two control antibodies were raised in female Balb/c mice againstpurified soluble MUC1 from CHO ldlD MUC1 cells grown in GalNAc (MAb5E10) or Gal and GalNAc, followed by neuraminidase treatment (MAb 1B9).Immunizations were performed by one subcutaneous injection of 40 μg/100μl of immunogen emulsified in Freund's complete adjuvant followed by twoinjections with Freund's incomplete adjuvant at 2-3 weeks intervals andfinally a boost without adjuvant. The two clones were selected byimmunocytochemistry as described above with different selectioncriteria. MAb 5E10 was selected since it reacted with all the testedMUC1 expressing cell lines independently of O-glycosylation andtherefore potentially could serve as a universal anti-MUC1 MAb. MAb 1B9was selected because it showed specificity for neuraminidase treatedcells presenting the T antigen.

ELISA-Assays

Enzyme-linked immunosorbent assays (ELISA) were performed usingNunc-Immuno MaxiSorp F96 plates (Nunc, Roskilde, Denmark).Unbiotinylated glycopeptides were serially diluted from an initialconcentration of 2 μg/ml and coated 1 h at 37° C. or over night at 4° C.in carbonate-bicarbonate buffer (pH 9.6). For capture ELISA, plates werecoated 1 h at 37° C. or over night at 4° C. with 1.5 μg/ml ofstreptavidin (Sigma-Aldrich, St. Louis, Mo.) in carbonate-bicarbonatebuffer (pH 9.6). Plates were blocked with SuperBlock Blocking Buffer(Pierce, Rockford, Ill.) for 1 h at room temperature. Thestreptavidin-coated plates were incubated with biotinylatedglycopeptides serially diluted from an initial concentration of 2 μg/mland incubated for 1 h at 37° C. or over night at 4° C. Subsequently,plates were incubated with monoclonal antibodies for 2 h at roomtemperature or over night at 4° C. 5E5, 2D9, 1B9, 5E10, and SM3 wereused as undiluted culture supernatants, whereas MY.1E12 ascites wereused 1:1000 and purified BW835 was used at 1 μg/ml. MY.1E12 was kindlyprovided by Dr. T. Irimura, and BW835 by Drs. F. -G. Hanisch and T.Schwientek. Sera from MUC1 transgenic mice immunized with Tn-MUC1 wereserially diluted in 2% BSA in PBS from an initial dilution of 1:100 or1:200. Bound antibodies were detected with HRP-conjugated polyclonalrabbit anti-mouse immunoglobulins (Dako, Glostrup, Denmark). Plates weredeveloped with TMB+ one-step substrate system (Dako, Glostrup, Denmark),reactions stopped with 1 N H₂SO₄, and read at 450 nm.

Immunocytochemistry

Cell lines were fixed for 10 min in ice-cold acetone. Fixed cells wereincubated overnight at 4° C. with undiluted MAb supernatants, followedby incubation for 45 min at room temperature with fluoresceinisothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulins(Dako, Glostrup, Denmark). Slides were mounted in glycerol containingp-phenylenediamine and examined in a Zeiss fluorescence microscope(FluoresScience, Hallbergmoos, Germany).

Results Generation of MUC1 Monoclonal Antibodies

The MAb 5E5 (IgG1) was raised against 60-mer MUC1 tandem repeat peptidecarrying 15 GalNAc residues conjugated to KLH as described previously(Sorensen et al. 2006). This antibody was shown to specifically reactwith MUC1 carrying Tn or STn in the tandem repeat domain and reacts withthe vast majority of breast carcinomas while showing no reactivity withnormal breast epithelia (Sorensen et al. 2006). 5E5 was originallyselected because its reactivity pattern essentially mirrored that oftotal sera from MUC1 transgenic mice immunized with MUC1 tandem repeatglycopeptides with complete Tn- or STn-glycosylation (Sorensen et al.2006). In the present study, we have reproduced the immunization andscreening protocol and isolated another monoclonal antibody, 2D9 (IgG1),which exhibits essentially the same specificity (FIG. 6), demonstratingthat such antibodies are prevalent.

Two additional MUC1 antibodies were raised against purified recombinantsecreted MUC1 (rMUC1) expressed in CHO ldlD cells grown in GalNAc toproduce the Tn glycoform (5E10) or grown in Gal and GalNAc to producethe ST glycoform, which after neuraminidase treatment was reduced to theT glycoform (1B9). By immunocytochemistry, MAb 5E10 reacted with all theMUC1 expressing cell lines tested and therefore potentially could serveas a universal anti-MUC1 MAb. MAb 1B9 was selected because it showedspecificity for neuraminidase treated cells presenting the T glycoformof MUC1 (data not shown).

Epitope Mapping of MAbs 5E5 and 2D9 Raised Against Tn-MUC1 Tandem RepeatGlycopeptides

The specificity of the antibodies was initially determined by directbinding ELISA assays using a panel of 60-mer glycopeptides produced bychemoenzymatic methods (FIG. 5) (Sorensen et al. 2006). The MAbs 5E5 and2D9 exhibited a similar reactivity pattern with high selectivity forMUC1 tandem repeat glycopeptides with Tn and STn O-glycans and bothantibodies showed preference for Tn-MUC1 glycoforms with highestO-glycan occupancy; however, in direct binding assays, 2D9 showedsignificantly better reactivity with Tn glycoforms compared to STnglycoforms (not shown). In order to fully assess the binding specificityof the antibodies and eliminate issues with differences in adsorptionand presentation of MUC1 peptides and glycopeptides in direct bindingELISA assays, a streptavidin-biotin capture ELISA was developed using alarge panel of 60-mer based MUC1 biotinylated glycopeptides (FIG. 5).The results shown in FIG. 6 clearly confirm that the two MAbs, 5E5 and2D9, react with a glycopeptide epitope where the glycan can be Tn orSTn, with at least two O-glycans and preferably three or five O-glycansper MUC1 repeat. This could suggest that an O-glycan in either the VTSA(SEQ ID NO. 6) or the GSTV (SEQ ID NO. 13) region of the sequence isrequired for the epitope (FIG. 5). Recombinant expression of MUC1 in CHOldlD cells allows presentation of different glycoforms of MUC1 (Sorensenet al. 2006). MAbs 5E5 and 2D9 reacted with Tn-MUC1 but not with T-MUC1glycoforms as predicted by the lack of reactivity with T and ST-MUC1glycopeptides (FIG. 6, panels A and D). Interestingly, weak reactivitywas observed with the core 3 O-glycosylated glycoform(GlcNAcβ1-3GalNAcα1-O-Ser/Thr) but only when this is presented withthree and not with five O-glycans per tandem repeat. The significance ofthis is not clear at present, and expression of the β3Gn-T6 enzymesynthesizing the core 3 O-glycan structure is limited to stomach, colon,and small intestine.

Further glycopeptide variants are required to more precisely define theepitopes; however, present enzymatic glycosylation of 60-mer MUC1peptides are limited by the substrate specificities of polypeptideGalNAc-transferases. Therefore, two 25-mer peptides with valinesubstitutions of selected threonine residues were used tochemoenzymatically produce glycoforms with Tn at individual sitesutilizing GalNAc-T11 (FIG. 5). Besides Tn-glycosylation of the initialThr, the two glycopeptides were either Tn-glycosylated at Thr in theVTSA (SEQ ID NO. 6) region (2Tn-TAP25V21) or at Thr in the GSTA (SEQ IDNO. 7) region (2Tn-TAP25V9). Enzymatic Tn-glycosylation of both Ser andThr in the GSTA (SEQ ID NO. 7) region, which was observed to increasereactivity with the biotinylated 60-mer peptide, was not possible withthe TAP25V9 peptide. As shown in FIG. 6 (Panels B and E) 5E5 and 2D9 didnot react with 2Tn-TAP25V21 with Tn-glycosylation at Thr in the VTSA(SEQ ID NO. 6) region, whereas strong reactivity was found with the2Tn-TAP25V9 glycopeptide with Tn-glycosylation at Thr in the GSTA (SEQID NO. 7) region. This reactivity pattern was confirmed in directbinding ELISA with a panel of synthetic MUC1 glycopeptides with onesingle Tn or T O-glycan (FIG. 5). 5E5 and 2D9 showed strong reactivitytowards the glycopeptide with Tn at the Thr in the GSTA (SEQ ID NO. 7)region, whereas no reactivity was seen when the T glycan was carried onthis threonine or with the other Tn- or T-glycosylated glycopeptides(FIG. 6, panels C and F). In summary, 5E5 and 2D9 reacted with MUC1glycopeptides when Thr in GSTA (SEQ ID NO. 7) is Tn- or STn-glycosylatedand stronger when both Ser and Thr are glycosylated.

Specificity Analysis of Total Serum from Tn-MUC1 Immunized MUC1Transgenic Mice

Total serum of mice immunized with the 15Tn-MUC1 60-mer glycopeptideconjugated to KLH showed the same preference for high-density Tn- andSTn-MUC1 glycopeptides (FIG. 7, panel A). More importantly, the samespecificity for the Tn-glycosylated GSTA (SEQ ID NO. 7) sequence wasobserved with the valine-substituted glycopeptides (FIG. 7, panel B).Taken together with the data above, these results clearly indicate thatthe GSTA (SEQ ID NO. 7) region of the MUC1 tandem repeat glycosylatedwith Tn and/or STn represents a novel immunodominant MUC1 glycopeptideepitope.

Characterization of MAbs 1B9 and 5E10

The two MAbs raised against recombinant MUC1 glycoprotein expressed inCHO ldlD cells were analyzed with the capture ELISA using the panel ofMUC1 60-mer biotinylated glycopeptides. MAb 1B9 showed strong reactivitywith the MUC1 glycopeptide with three T O-glycans per tandem repeat,whereas only an extremely weak reaction was observed with theglycopeptide fully substituted with five T O-glycans per tandem repeat(FIG. 8, panel A). Intermediate reactivity was seen with peptidessubstituted with core 3 and ST, but only with peptides with threeglycans per tandem repeat (FIG. 8, panel A). ELISA with MUC1glycopeptides with one single T O-glycan showed reactivity with thepeptide T-glycosylated at Thr in the amino acid sequence GSTA (SEQ IDNO. 7), but also, although weaker, with the peptide T-glycosylated atThr in the amino acid sequence VTSA (SEQ ID NO. 6) (FIG. 8, panel B). Noreactivity was seen with the MUC1 glycopeptides with one single TnO-glycan. Furthermore, 1B9 did not react with CHO ldlD MUC1-expressingcells when grown in GalNAc alone, but only when Gal is added to thegrowth medium, leading to expression of ST-MUC1. Significantly enhancedreactivity was seen after neuraminidase treatment of the cells to exposeT-MUC1 (FIG. 8, panel C). These data suggest that the epitope for 1B9 isnot a glycopeptide epitope, but rather a conformational epitoperequiring glycosylation with β1-3 linked disaccharides (T or core 3) ineither the VTSA (SEQ ID NO. 6) or the GSTA (SEQ ID NO. 7) regions, butnot the PDTR (SEQ ID NO. 4) region.

The MAb 5E10 showed highest reactivity with biotinylated MUC1 60-merpeptide or when the peptide is either unglycosylated or substituted withonly two Tn glycans per tandem repeat. Reactivity towardsTn-glycosylation decreased with increasing density of glycosylation. Anadditional decrease in reactivity was seen with the introduction ofT-glycosylation, followed by a further decrease by introduction of core3 glycosylation. Lowest reactivity was seen with increasing degrees ofsialylation, especially when NeuAc is α2-6-linked to GalNAc (STn). Noreactivity at all was seen with the peptide fully substituted with STn(FIG. 9, panel A). In ELISA with biotinylated valine-substituted invitro Tn-glycosylated MUC1 peptides, equal reactivity was seen with allfour peptides regardless of glycosylation (FIG. 9, panel B). In ELISAwith MUC1 glycopeptides with one single Tn or T O-glycan, equalreactivity was seen with all peptides except for the peptideT-glycosylated at Thr in the amino acid sequence GSTA (SEQ ID NO. 7)(FIG. 9, panel C). In immunocytology, 5E10 reacted with CHO ldlDMUC1-expressing cells independently on co-culturing with GalNAc, Gal, orno sugar at all (data not shown). In summary, 5E10 reacted with all MUC1glycoforms tested with the exception of complete STn occupancy.

Comparison with Other MAbs Previously Reported to React with MUC1Glycoforms

The MAb SM3 binds the PDTR region of the MUC1 tandem repeat andTn-glycosylation of the Thr enhances its binding. In agreement withprevious reports, SM3 preferentially reacted with unglycosylated peptideand the glycopeptides with complete O-glycan occupancy of five O-glycansper tandem repeat, while reactivity with glycopeptides with two andthree O-glycans was lower (FIG. 9, panel D). These results confirm andextend our previous studies to demonstrate that T, ST and core 3O-glycans react equally well. For this study we did not have core 2glycoforms, but studies with cell lines clearly indicate that core 2glycosylation of MUC1 blocks the SM3 epitope. Little reactivity wasobserved with the biotinylated valine-substituted peptides, whetherunglycosylated or with Tn-glycosylation of Thr in the amino acidsequences VTSA (SEQ ID NO. 6) or GSTA (SEQ ID NO. 7) (FIG. 9, panel E).In ELISA with MUC1 glycopeptides with one single Tn or T O-glycan, highreactivity was seen when Thr in the amino acid sequence PDTR (SEQ ID NO.4) is substituted with either T or Tn. Lower reactivity was seen withthe remaining T-MUC1 glycopeptides, whereas hardly any reactivity wasobserved with the remaining Tn-MUC1 glycopeptides (FIG. 9, panel F).

MAb BW835 reacted in the capture ELISA with biotinylated MUC1 60-merglycopeptides fully glycosylated with the disaccharides T (FIG. 8, panelC). Interestingly, BW835 reacted equally well with the core 3O-glycosylated peptide indicating that the antibody does not require theT glycoform per se. Lower reactivity was also found with the fullyST-glycosylated peptide. Weak reactivity was observed with the Tglycopeptide with only three O-glycans, and similar weak reactivity wasfound with the fully Tn-glycosylated glycopeptide. No reactivity wasfound with glycopeptides carrying two or three Tn glycans per TR,STn-glycosylated, or unglycosylated peptides. These results are inagreement with and extend previous characterization of the epitope.

In accordance with earlier published data, MY.1E12 showed strictspecificity for the ST-glycoforms of MUC1 as evaluated with biotinylated60-mer glycopeptides (FIG. 8, panel D). In contrast to BW835, MY.1E12showed preference for the peptide with three ST O-glycans per tandemrepeat, suggesting that the epitope is at least partially destroyed whenboth Thr and Ser in the VTSA (SEQ ID NO:6) region are glycosylated.

Example 3 Materials and Methods

A MUC1 60-mer peptide (VTSAPDTRPAPGSTAPPAHG)_(n=3) (SEQ ID NO. 1)representing three tandem repeats is glycosylated in vitro with 5 molesof Tn, STn, and T as described in Examples 1 and 2. Controlglycopeptides includes the MUC2 33-mer peptide(PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC) (SEQ ID NO. 14) with the sameglycoforms. Murine monoclonal anti-MUC1 antibodies, 5E10, 5E5 and 1B9,were described in Example 2. Murine monoclonal antibodies to Tn (3E1,5F4), T (3F1, TKH2) and T (HHB, 3C9) are produced as previouslydescribed (Kjeldsen et al, 1989; Kjeldsen et al, 1988; Hirohashi et al,1985; Clausen et al, 1988). Monosaccharides GalNAc, GlcNAc, Gal, Glc andNeuAc are bought from Sigma, GalNAcα-agarose (GlycoSorb-1) is boughtfrom GlycoRex. OSM and asialo-OSM are prepared as previously described(Reis et al, 1998b; Reis et al, 1998a). BSM is bought from Sigma andasialo-BSM prepared as previously described by neuraminidase treatment(Reis et al, 1998b; Reis et al, 1998a). Tn (GalNAcα1-), STn(NeuAcα2-6GalNAcα1-) and T (Galβ1-3GalNAcα1-) polyvalent PAA conjugatesare bought from GlycoTech.

ELISA-Assays

Enzyme-linked immunosorbent assays (ELISA) are performed usingNunc-Immuno MaxiSorp F96 plates (Nunc, Roskilde, Denmark). Peptides andglycopeptides are coated at concentrations of 1, 0.2, and 0.05 μg/ml for1 h at 37° C. or over night at 4° C. in carbonate-bicarbonate buffer (pH9.6). Plates are blocked with SuperBlock Blocking Buffer (Pierce,Rockford, Ill.) for 1 h at room temperature. Subsequently, plates areincubated with dilutions of monoclonal anti-MUC1 antibodies 5E5, 1B9,and 5E10 and anti-carbohydrate antibodies 3E1, 5F4, 3F1, TKH2, HHB, and3C9 (starting from undiluted culture supernatants) for 2 h at roomtemperature. In subsequent inhibition experiments fixed concentrationsof (glyco)peptides and monoclonal antibodies are used, and theappropriate dilution of monoclonal antibodies are preincubated withserially diluted inhibitor carbohydrates and glycoconjugates (startingfrom 0.5M for monosaccharides, 10 μg/ml (glyco)peptides, 100 μg/ml forPAA conjugates and 10 μg/ml mucins) for 30 min at room temperature,before transfer to coated ELISA plates. Bound antibodies are detectedwith HRP-conjugated polyclonal rabbit anti-mouse immunoglobulins (Dako,Glostrup, Denmark). Plates are developed with TMB+ one-step substratesystem (Dako, Glostrup, Denmark), reactions stopped with 1 N H2504, andread at 450 nm.

Results Determination of End-Point Titer of Antibodies

Initial ELISA assays are performed to define appropriate coatingconcentrations of peptides and glycopeptides and appropriate dilutionsof antibodies for further inhibition assays. For each antibodyappropriate antigen and antibody dilutions are determined by evaluationof end-point titer and conditions yielding OD450 readings ofapproximately 1 are used for further studies.

Inhibition of antibody binding to glycopeptides with Tn, STn and Tglycosylation—Antibodies to carbohydrate haptens—The antibodies reactivewith Tn, STn and T irrespective of the peptide backbone of glycopeptidesreacts with both MUC1 and MUC2 glycopeptides with the respectiveglycoforms.

Thus, anti-Tn antibodies 1E3 and 5F4 reacts only with theTn-glycopeptides, anti-STn antibodies TKH2 and 3F1 reacts only with theSTn-glycopeptides, while anti-T antibodies react with T-glycopeptides.Inhibition ELISA assays further demonstrate that binding to therespective glycopeptides can be inhibited by the correspondingglycopeptides, mucins (Tn antibodies with asialo-OSM, STn antibodieswith OSM and T antibodies with asialo-BSM, PAA conjugates, as well aswith high concentrations of monosaccharides (Tn antibodies with GalNAc,STn antibodies with NeuAc, and T antibodies with Gal). Furthermore, theGlycoSorb-1 Tn adsorbant can inhibit the Tn antibodies.

Antibodies to MUC1—The antibody reactive with the MUC1 peptide (5E10) isinhibited by all MUC1 peptides and glycopeptides, whereas MUC2 peptidesand glycopeptides as well as all other glycans and glycoconjugates cannot inhibit. In striking contrast, the MUC1 Tn/STn glycoform specificantibodies 5E5 and 2D9 are inhibited only by Tn-MUC1 glycopeptides andto a much lesser degree by STn-MUC1 glycopeptides. Similarly, the MUC1 Tglycoform specific antibody 1B9 is only inhibited by the T-MUC1glycopeptides.

REFERENCES

Burchell, J. M., Mungul, A. & Taylor-Papadimitriou, J. (2001). O-linkedglycosylation in the mammary gland: changes that occur duringmalignancy. J. Mammary. Gland. Biol. Neoplasia., 6, 355-364.

Clausen, H., Stroud, M., Parker, J., Springer, G., & Hakomori, S. (1988)Monoclonal-Antibodies Directed to the Blood Group-A AssociatedStructure, Galactosyl-A-Specificity and Relation to theThomsen-Friedenreich Antigen. Molecular Immunology, 25, 199-204.

Hanisch, F. G., Stadie, T. & Bosslet, K. (1995). Monoclonal antibodyBW835 defines a site-specific Thomsen-Friedenreich disaccharide linkedto threonine within the VTSA motif of MUC1 tandem repeats. Cancer Res.,55, 4036-4040.

Kjeldsen, T., Clausen, H., Hirohashi, S., Ogawa, T., Iijima, H., &Hakomori, S. (1988) Preparation and Characterization ofMonoclonal-Antibodies Directed to the Tumor-Associated O-LinkedSialosyl-2-]6 Alpha-N-Acetylgalactosaminyl (Sialosyl-Tn) Epitope. CancerResearch, 48, 2214-2220.

Kjeldsen, T., Hakomori, S., Springer, G. F., Desai, P., Harris, T., &Clausen, H. (1989) Coexpression of Sialosyl-Tn(Neuac-Alpha-2-]6Galnac-Alpha-1-]O-Ser/Thr) and Tn(Galnac-Alpha-1-]O-Ser/Thr) Blood-Group Antigens on Tn Erythrocytes. VoxSanguinis, 57, 81-87.

Mensdorff-Pouilly, S., Petrakou, E., Kenemans, P., van Uffelen, K.,Verstraeten, A. A., Snijdewint, F. G. M., van Kamp, G. J., Schol, D. J.,Reis, C. A., Price, M. R., Livingston, P. O., & Hilgers, J. (2000)Reactivity of natural and induced human antibodies to MUC1 mucin withMUC1 peptides and N-acetylgalactosamine (GalNAc) peptides. InternationalJournal of Cancer, 86, 702-712.

Reis, C. A., Hassan, H., Bennett, E. P., & Clausen, H. (1998a)Characterization of a panel of monoclonal antibodies using GalNAcglycosylated peptides and recombinant MUC1. Tumor Biology, 19, 127-133.

Reis, C. A., Sorensen, T., Mandel, U., David, L., Mirgorodskaya, E.,Roepstorff, P., Kihlberg, J., Hansen, J. E. S., & Clausen, H. (1998b)Development and characterization of an antibody directed to analpha-N-acetyl-D-galactosamine glycosylated MUC2 peptide. GlycoconjugateJournal, 15, 51-62.

Sorensen, A. L., Reis, C. A., Tarp, M. A., Mandel, U., Ramachandran, K.,Sankaranarayanan, V., Schwientek, T., Graham, R., Taylor-Papadimitriou,J., Hollingsworth, M. A., Burchell, J. & Clausen, H. (2006).Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptideselicit cancer-specific anti-MUC1 antibody responses and overridetolerance. Glycobiology, 16, 96-107.

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Takeuchi, H., Kato, K., da-Nagai, K., Hanisch, F. G., Clausen, H. &Irimura, T. (2002). The epitope recognized by the unique anti-MUC1monoclonal antibody MY.1E12 involves sialyl alpha 2-3galactosyl beta1-3N-acetylgalactosaminide linked to a distinct threonine residue in theMUC1 tandem repeat. J. Immunol. Methods, 270, 199-209.

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1. A method of inducing a cancer specific immune response toward MUC1comprising immunization of an animal with an immunogenic glycopeptidecomprising a GSTA motif, wherein said GSTA motif is O-glycosylated atleast at the T-residue or at the S-residue of the GSTA motif.
 2. Themethod of claim 1, wherein the immune response toward MUC1 is eitherinnate immunity, humoral immunity, cellular immunity or any combinationsthereof.
 3. The method according to claim 1, wherein said MUC1 isaberrantly glycosylated and expressed on cancer cells.
 4. The methodaccording to claim 1, wherein the O-glycosylation is selected from thegroup consisting of a STn glycan and a Tn glycan.
 5. The methodaccording to claim 1, wherein both the S-residue and T-residue isO-glycosylated.
 6. The method according to claim 1, wherein theO-glycosylation is either STn glycan or Tn glycan.
 7. The methodaccording to claim 1, wherein the GSTA motif is present in a tandemrepeat of 20 amino acid residues, said tandem repeat comprising fivepotential sites for O-glycosylation.
 8. The method according to claim 1,wherein the GSTA motif is present in a tandem repeat, wherein thesequence of the tandem repeat is selected from the group consisting of:a) VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 1); b) naturally occurring variantsof SEQ ID NO: 1 with at least 75% similarity to SEQ ID NO:1; c)artificial variants of SEQ ID NO:1 wherein said artificial variants areprepared by one or more conservative substitutions and wherein saidartificial variants have at least 75% similarity to SEQ ID NO: 1; and d)truncated fragments of SEQ ID NO: 1 with 1-3 deleted amino acids.
 9. Themethod according to claim 1, wherein the GSTA motif is present in atandem repeat, and wherein the glycosylation pattern of the tandemrepeat is selected from the group consisting of:VT^(Tn)SAPDTRPAPGST^(Tn)APPAHG; VT^(Tn)SAPDTRPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)SAPDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)S^(Tn)APDTRPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)S^(Tn)APDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG;VT^(STn)SAPDTRPAPGST^(STn)APPAHG;VT^(STn)SAPDTRPAPGS^(STn)T^(STn)APPAHG;VT^(STn)SAPDT^(STn)RPAPGS^(STn)T^(STn)APPAHG;VT^(STn)S^(STn)APDTRPAGS^(STn)T^(STn)APPAHG; andVT^(STn)S^(STn)APDT^(STn)RPAPGS^(STn)T^(STn)APPAHG.


10. The method according to claim 1, wherein the immunogenicglycopeptide comprises more than two tandem repeats.
 11. The methodaccording to claim 1, wherein the immunogenic glycopeptide comprisesmore than one tandem repeat.
 12. The method according to claim 1,wherein the immunogenic glycopeptide is coupled to a carrier selectedfrom the group consisting of: human serum albumin, keyhole limpethemocyanin (KLH), thyroglobulin, ovalbumin, influenza hemagglutinin,PADRE polypeptide, malaria circumsporozite (CS) protein, hepatitis Bsurface antigen (HBSAgI9-2s), Heat Shock Protein (HSP) 65, Mycobacteriumtuberculosis, cholera toxin, cholera toxin mutants with reducedtoxicity, diphtheria toxin, CRM 97 protein that is cross-reactive withdiphtheria toxin, recombinant Streptococcal C5a peptidase, Streptococcuspyogenes ORF1224, Streptococcus pyogenes ORF1664, Streptococcus pyogenesORF2452, Chlamydia pneumonia ORF T367, Chlamydia pneumoniae ORF T858,Tetanus toxoid and HIVgp120T1.
 13. The method according to claim 1,wherein the mammal is selected from the group consisting of: a human, amouse, a rat, a rabbit, a sheep, a goat, and a dog.
 14. A compositioncomprising an immunogenic glycopeptide comprising a GSTA motif, whereinsaid GSTA motif is O-glycosylated at least at the T-residue or at theS-residue of the GSTA motif.
 15. The composition according to claim 14,wherein the GSTA motif is present in a tandem repeat, and wherein theglycosylation pattern of the tandem repeat is selected from the groupconsisting of: VT^(Tn)SAPDTRPAPGST^(Tn)APPAHG;VT^(Tn)SAPDTRPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)SAPDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)S^(Tn)APDTRPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)S^(Tn)APDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG;VT^(STn)SAPDTRPAPGST^(STn)APPAHG;VT^(STn)SAPDTRPAPGS^(STn)T^(STn)APPAHG;VT^(STn)SAPDT^(STn)RPAPGS^(STn)T^(STn)APPAHG;VT^(STn)S^(STn)APDTRPAGS^(STn)T^(STn)APPAHG; andVT^(STn)S^(STn)APDT^(STn)RPAPGS^(STn)T^(STn)APPAHG.


16. The composition according to claim 14, said composition being apharmaceutical composition.
 17. The composition according to claim 14,wherein the composition is a cancer vaccine for treatment or preventionof breast cancer, ovarian cancer, pancreatic cancer, or lung cancer. 18.A method of treating or preventing cancer comprising administrating apharmaceutical composition comprising an immunogenic glycopeptidecomprising a GSTA motif, wherein said GSTA motif is O-glycosylated atleast at the T-residue or at the S-residue of the GSTA motif.
 19. Themethod according to claim 18, wherein the GSTA motif is present in atandem repeat, and wherein the glycosylation pattern of the tandemrepeat is selected from the group consisting of:VT^(Tn)SAPDTRPAPGST^(Tn)APPAHG; VT^(Tn)SAPDTRPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)SAPDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)S^(Tn)APDTRPAPGS^(Tn)T^(Tn)APPAHG;VT^(Tn)S^(Tn)APDT^(Tn)RPAPGS^(Tn)T^(Tn)APPAHG;VT^(STn)SAPDTRPAPGST^(STn)APPAHG;VT^(STn)SAPDTRPAPGS^(STn)T^(STn)APPAHG;VT^(STn)SAPDT^(STn)RPAPGS^(STn)T^(STn)APPAHG;VT^(STn)S^(STn)APDTRPAGS^(STn)T^(STn)APPAHG; andVT^(STn)S^(STn)APDT^(STn)RPAPGS^(STn)T^(STn)APPAHG.